The traditional view of the nucleus has changed substantially in recent years. Today, it is recognized that many of the processes associated with gene expression are localized within a very complex, dynamic nuclear framework. How macromolecular complexes important for RNA splicing are assembled within the nucleus is an important focus area in biology. Errors in splicing have been linked to an array of human neurodegenerative diseases and tumorigenesis underscoring the high level of specificity required for RNA processing events. Recently, it has become clear that protein factors play a key role in determining splicesome assembly and splice site selection. One class of splicing factors known as SR proteins for their unusual arginine-serine dipeptide repeats regulates splicing at several stages. These factors become multiply phosphorylated by SR protein kinases (SRPKs), modifications that have profound effects on protein function within the splicesome. Despite the biological significance of phosphorylation, little is known about how the SR proteins become phosphorylated and how specific phosphorylation events or patterns are linked to the control of splicing. The goals of this proposal will include the identification of the serine phosphorylation sites in the SR protein ASF/SF2, a substrate for human SRPK1. How these serines get phosphorylated will be studied using site replacement mutants, autoradiography and mass spectrometry. The importance of these serines for the control of splicing will be addressed in large macromolecular complexes using an in vitro splicing assay. Factors which initiate phosphorylation will be studied using deletion mutants and substrate chimeras. While all SRPKs are constitutively active, it is likely that the structural mechanism of activation is species specific. The origins of this phenomenon will be addressed using mutagenesis and X-ray crystallography. The experiments outlined in this proposal are designed to fully characterize a novel class of splicing factors and their enzymatic regulators.